Study design, duration and site
A hospital-based cross-sectional study was carried out from 20th June to December 2018, among the clinically suspected Urinary Tract Infected (UTI) patients visiting the hospital. The clinical sample processing followed by identification of uropathogens were carried out in Microbiology Laboratory of Kathmandu Model Hospital, Kathmandu, while the nucleic acid extraction and PCR amplification were done in Department of Microbiology, GoldenGate International College, Kathmandu.
Inclusion and exclusion criteria
This study included the patients of both sexes and all age group attending the hospital with suspicion of UTI infection. The samples which were adequately collected and properly labeled were included in the study. Those samples which were not collected with standard collection procedure, inadequately collected, improperly labeled and samples with visible contamination were excluded in the study. A repeated sample was requested in such cases. The urine samples from the patients whose health status was not mentioned as the diabetic and non-diabetics and those who were taking antibiotic less than 24 hours before visiting hospital were also excluded.
Study variables
The study variables included were age, sex, out-patients, inpatients department, health status including diabetic or non-diabetic and history of antibiotic taking within 24 hours of visiting hospital using standard data form assigned by the hospital.
Sample size
Consent for participation was collected from all participants during enrolment and before data and sample collection. UTI patients or suspected patients were instructed to collect mid-stream urine sample in sterile, clean and leakproof vials. A total of 1267 non-duplicate mid-stream Urine (MSU) specimens were obtained and processed immediately in the Microbiology laboratory of Kathmandu Model Hospital. The adequately collected samples from both sexes and all age groups were included while the improperly collected and poorly labelled samples with visible contamination were excluded for the study. They were requested to repeat the sample collection. Besides demographic data, medical status of patients (diabetic or non-diabetic and inpatients or outpatients) had also been recorded.
Sample processing
The urine samples were cultured onto the Cysteine Lactose Electrolyte Deficient (CLED) agar by the semi-quantitative culture technique using a standard calibrated loop [17]. The agar plates were incubated at 370C for overnight. The bacteria developed in the pure culture with a load greater than 105 CFU/ml were considered as significant growth and included in this study. The bacteria were identified by standard microbiological procedures including microscopy, colony microbiology and biochemical tests as described by American Society of Microbiology (ASM). Among different bacterial isolates, only E. coli isolates were further processed for molecular studies [18].
Antimicrobial susceptibility testing
Susceptibility tests of the bacterial isolates towards various antibiotics were performed by the modified Kirby-Bauer disc diffusion method using Mueller Hinton Agar. The E. coli (ATCC 25922) was used as the control strains. Antibiotics Amoxicillin (10 µg), Cefexime (5 µg), Cotrimoxazole (25 µg), Cefotaxime (30 µg), Ceftazidime (30 µg), Ceftriaxone (30 µg), Gentamicin (10 µg), Levofloxacin (5 µg), Ofloxacin (5 µg), Norfloxacin (5 µg) and Nitrofurantoin (300 µg) were used in the primary testing while for MDR isolates Amikacin (30 µg), Amoxicillin-clavulanic acid (20/10 µg), Cefoperazone/Sulbactam (75/30 µg), Cefepime (50 µg), Doxycycline (30 µg), Imipenem (10 µg), Meropenem (10 µg), and Piperacillin/Tazobactam (100/10 µg) were used for further testing. Further Polymyxin B (300 µg), Colistin (10 µg) and Tigecycline (15 µg) was used for 14 bacterial isolates [19]. Subsequently, the rate MDR E. coli was determined [20].
Phenotypic characterization of the ESBL producers
The E. coli isolates were screened for possible ESBL production using Ceftazidime (30 µg) and Cefotaxime (30 µg). The suspected ESBL producing E. coli were subjected to Combined Disk (CD) assay using Ceftazidime (30 µg) with Ceftazidime plus Clavulanic acid (30/10 µg) and Cefotaxime (30 µg) with Cefotaxime plus Clavulanic acid (30/10 µg) for phenotypic confirmation [19].
Molecular characterization of blaTEM and blaCTX−M genes: The plasmid DNA was extracted from phenotypically confirmed ESBL producing E. coli by the alkaline lysis method followed by the phenol: chloroform purification method [21, 22]. A conventional linear PCR was used to amplify the blaTEM and blaCTX−M genes in the extracted plasmid DNA. The blaTEM gene was amplified by using a primer with forward nucleotide sequence 5’-GAGACAATAAGGGTGGTAAAT-3’ and reverse nucleotide sequence 5’- AGAAGTAAGTTGGCAGCAGTG-3’ [23]. Similarly, the blaCTX−M gene was amplified by using a primer with forward nucleotide sequence 5’-TTTGCGATGTGCAGTACCAGTAA-3’ and reverse nucleotide sequence 5’-CTCCGCCTGCCGGTTTTAT-3’. The master mix containing 200 µM of dNTPs, 0.5 U/µl of Taq polymerase in 1X PCR buffer and 25 mM MgCl2 from Qiagen was used [24]. The PCR was carried out in 25 µl volume which was prepared by mixing the 13 µl of the master mix, 8 µl of the double-distilled water, 0.5 µl each of the forward and reverse primer and 3 µl of the template DNA. Amplification reactions were carried out using the reaction conditions: initial denaturation at 950C for 15 minutes; followed by 35 cycles of denaturation at 940C for 45 seconds, annealing at 550C for blaTEM genes and 560C for the blaCTX−M gene for 30 seconds, extension at 720C for 3 minutes and a final extension at 720C for 10 minutes. The PCR products were analyzed on 1.5% agarose gel electrophoresis with 0.2 µg/ml concentration of ethidium bromide and then visualized by the UV-trans illuminator. The protocol from Sharma et al., and Edelstein et al., were followed with slight modification [23, 24].
Quality control:
E. coli ATCC 35218 and E. coli ATCC 25922, obtained from Central Department of Microbiology, TU were used as control strains for screening of blaTEM and blaCTX−M genes.
Data analysis
All the data were analyzed by the Statistical Package for Social Science (SPSS) software (Version 22.0) and MS Excel (Version 10).